1. Field of the Invention
The invention relates to methods of detecting bacterial strains associated with sludge problems at wastewater treatment plants. More specifically, the invention relates to the identification of novel bacterial strains associated with sludge problems at wastewater treatment plants. Most specifically, the invention relates to nucleic acid probes for use in detecting bacterial strains associated with sludge problems.
2. Background Art
Two important desired qualities of sludges at wastewater treatment plants are dewaterability and compactability. Microscopic observations at industrial wastewater treatment facilities have demonstrated that the presence of zoogloeal clusters is correlated with episodes of poor sludge dewaterability, and the presence of Hyphomicrobium sp. is associated with poor sludge compaction.
Plant operations designed to enhance endogenous respiration in one of three aeration basins have resulted in significant decreases in sludge yield (Bullard and Barber, 1994). However, periodic episodes of poor sludge dewaterability have limited applications of this sludge minimization strategy (Barber et al., 1995). Microscopy has indicated that decreased dewatering potential is correlated with the presence of amorphous zoogloeal clusters, although these clusters are also commonly present at lesser abundance during periods of good sludge quality.
Identifying and monitoring organisms forming zoogloeal clusters in activated sludge is complicated due to the lack of distinctive morphological or physiological characteristics delineating the genus Zoogloea (Unz, 1984). Historically, identification of species as Zoogloea spp. has been based primarily on the fact that they were isolated from wastewater and formed flocs (Crabtree and McCoy, 1967; Unz, 1971; Friedman and Dugan, 1968). The lack of distinctive conserved characteristics has rendered microscopic observation the only currently available monitoring method.
New developments in molecular based methods for microbial classification and identification, based on the sequence of the RNA found in the small ribosomal subunit (16S rRNA), have made possible a more complete and definitive analysis of complex microbial communities. However, this technology has not been fully utilized in the analysis of sludge microbial communities.
16S rRNA sequence analysis of strains previously classified as Zoogloea spp. has indicated that these organisms are not closely related (Shin et al., 1993). Whereas Z. ramigera ATCC 19623 is in the alpha subclass of the Proteobacteria, Z. ramigera ATCC 19544 and Z. ramigera 25935 are members of the beta subclass. Probing of activated sludge with 16S rRNA probes developed for the three major type strains (Zoogloea spp. ATCC 19623, 19544, and 25935) has suggested that only Z. ramigera ATCC 19544 is commonly present as zoogloeal clusters (Rossello-Mora et al., 1995; Wagner et al., 1995). Whether additional species are involved in zoogloea formation and associated dewatering problems has not been determined.
The application of fluorescent probes for in situ detection of Gram-negative filamentous bacteria in activated sludge is relatively new (Bradford et al., 1997, Reyes et al., 1997, Wagner et al., 1993, Wagner et al., 1994). Although, these researchers have developed a variety of oligonucleotide probes for Proteobacteria, 16S rDNA probes for Hyphomicrobium spp. have not been previously reported. Hyphomicrobium spp. have been observed in sewage treatment plants (Holm et al., 1996) and their presence in soils, groundwater, freshwater ponds and lakes, marine samples and hypersaline lakes have also been reported. Previous studies have shown that Hyphomicrobium vulgare characteristically grows in clumps, often adhering to the walls of the culture vessel. Hyphomicrobium-like bacterium are commonly present in the natural environment, and utilized methanol under denitrifying conditions (Amaral et al., 1995). Hyphomicrobium spp. are competitive under low nutrient conditions (Bergey's Manual of Determinative Bacteriology).
The present invention provides the identity of organisms comprising zoogloeal clusters in a wastewater treatment plant and 16S rRNA oligonucleotide probes for their routine monitoring. The invention also provides 16S rRNA oligonucleotide probes for the routine monitoring of Hyphomicrobium spp.